Defective KIM-1 phagocytosis does not predispose to acute graft dysfunction after kidney transplantation in humans
Authors: Rita S. Suri, Ji Yun Lee, Matthew R. Ban, Bradly Shrum, Elena Tutunea-Fatan, Ola Z. Ismail, Stephen H. Leckie, Adam D. McIntyre, Qingyong Xu, Seung Ho Lee, Sonali N. de Chickera, Robert A. Hegele, Lakshman Gunaratnam
Journal: Kidney International (2022)

Ischemia-reperfusion injury (IRI) is common to several pathologic conditions, including myocardial infarction, stroke, acute kidney injury, and delayed graft function after kidney transplantation.1Eltzschig H.K. Eckle T. Ischemia and reperfusion--from mechanism to translation.Nat Med. 2011; 17: 1391-1401Crossref PubMed Scopus (2071) Google Scholar Clearance of apoptotic and necrotic cells by resident immune or epithelial cellsS1,S2 prevents release of harmful intracellular proinflammatory mediators, and is thought to represent an important mechanism to dampen secondary tissue damage and promote repair after acute injury, including IRI.2Arai S. Kitada K. Yamazaki T. et al.Apoptosis inhibitor of macrophage protein enhances intraluminal debris clearance and ameliorates acute kidney injury in mice.Nat Med. 2016; 22: 183-193Crossref PubMed Scopus (127) Google Scholar,3Lee J.Y. Ismail O.Z. Zhang X. et al.Donor kidney injury molecule-1 promotes graft recovery by regulating systemic necroinflammation.Am J Transplant. 2018; 18: 2021-2028Crossref PubMed Scopus (11) Google Scholar,S3,S4 In the kidney, proximal tubular epithelial cells (PTECs) are highly susceptible to apoptosis and necrosis after IRI.S5,S6 Phagocytosis of dead cells after kidney IRI is critically dependent on neighboring PTECs and kidney injury molecule-1 (KIM-1).2Arai S. Kitada K. Yamazaki T. et al.Apoptosis inhibitor of macrophage protein enhances intraluminal debris clearance and ameliorates acute kidney injury in mice.Nat Med. 2016; 22: 183-193Crossref PubMed Scopus (127) Google Scholar,4Ismail O. Zhang X. Bonventre J.V. et al.G protein, 12 (Galpha12) is a negative regulator of kidney injury molecule-1-mediated efferocytosis.Am J Physiol Renal Physiol. 2016; 310: F607-F620Crossref PubMed Scopus (16) Google Scholar KIM-1 is a glycoprotein upregulated on the apical surface of PTECs during acute injury of both native and transplanted kidneys.5Schroppel B. Kruger B. Walsh L. et al.Tubular expression of KIM-1 does not predict delayed function after transplantation.J Am Soc Nephrol. 2010; 21: 536-542Crossref PubMed Scopus (55) Google Scholar,S7,S8 Binding of KIM-1 to phosphatidylserine on apoptotic cells directly enables their clearance by PTECs, whereas clearance of necrotic cells requires KIM-1 interaction with an opsonin, apoptosis inhibitor of macrophage (AIM) protein.2Arai S. Kitada K. Yamazaki T. et al.Apoptosis inhibitor of macrophage protein enhances intraluminal debris clearance and ameliorates acute kidney injury in mice.Nat Med. 2016; 22: 183-193Crossref PubMed Scopus (127) Google Scholar,6Ichimura T. Asseldonk E.J. Humphreys B.D. et al.Kidney injury molecule-1 is a phosphatidylserine receptor that confers a phagocytic phenotype on epithelial cells.J Clin Invest. 2008; 118: 1657-1668Crossref PubMed Scopus (550) Google Scholar,S9 We previously observed significantly worse tissue injury and kidney dysfunction after kidney IRI in KIM-1–deficient compared with wild-type mice,7Ismail O.Z. Zhang X. Wei J. et al.Kidney injury molecule-1 protects against Galpha12 activation and tissue damage in renal ischemia-reperfusion injury.Am J Pathol. 2015; 185: 1207-1215Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar and similarly in mice transplanted with syngeneic KIM-1–deficient versus wild-type donor kidneys.3Lee J.Y. Ismail O.Z. Zhang X. et al.Donor kidney injury molecule-1 promotes graft recovery by regulating systemic necroinflammation.Am J Transplant. 2018; 18: 2021-2028Crossref PubMed Scopus (11) Google Scholar KIM-1–dependent phagocytosis is thus crucial for reducing kidney dysfunction after kidney IRI in mice,3Lee J.Y. Ismail O.Z. Zhang X. et al.Donor kidney injury molecule-1 promotes graft recovery by regulating systemic necroinflammation.Am J Transplant. 2018; 18: 2021-2028Crossref PubMed Scopus (11) Google Scholar but whether this is true in humans is unknown. HAVCR1, the gene for KIM-1, maps on chromosome 5q33.2 and is highly polymorphic.8Nakajima T. Wooding S. Satta Y. et al.Evidence for natural selection in the HAVCR1 gene: high degree of amino-acid variability in the mucin domain of human HAVCR1 protein.Genes Immun. 2005; 6: 398-406Crossref PubMed Scopus (31) Google Scholar Most HAVCRI coding region variants are concentrated within exon 4, which codes for the mucin domain. These variations may be driven by natural selection as hepatic KIM-1 is the receptor for hepatitis A virus, and this region is required for virus uncoating.S10,S11 It has been shown that deletion of the KIM-1 mucin domain drastically impairs phagocytosis by PTECs and worsens kidney dysfunction after kidney IRI in mice,9Yang L. Brooks C.R. Xiao S. et al.KIM-1-mediated phagocytosis reduces acute injury to the kidney.J Clin Invest. 2015; 125: 1620-1636Crossref PubMed Scopus (196) Google Scholar but the significance of dysfunctional KIM-1 on kidney outcomes in humans has not been examined. Delayed graft function (DGF), defined as need for dialysis within 7 days of kidney transplantation, is most commonly due to kidney IRI.S12 On the basis of the demonstrated importance of the KIM-1 mucin domain in mice, we hypothesized that naturally occurring functional polymorphisms within exon 4 of HAVCR1 would impair KIM-1–mediated phagocytosis, thereby predisposing to DGF in humans. Of >9000 known HAVCR1 polymorphisms, we identified exactly 3 within the mucin domain that result in amino acid change (nonsynonymous) and were common (allele frequency, >0.2; Supplementary Table S1). We tested our hypothesis in 2 parts. We first generated and expressed each of these variants in human embryonic kidney (HEK293) cells (which do not express endogenous KIM-1) and compared their capacity to mediate phagocytosis of apoptotic and necrotic cells compared with wild-type (hereafter referred to as "referent") KIM-1. We then performed HAVCR1 genotyping on >600 deceased kidney donors for these variants, and assessed whether receiving a donor kidney homozygous for the KIM-1 variant exhibiting the most severe dysfunction in vitro was associated with increased risk of DGF in recipients (see Supplementary Methods for details of experiments, genotyping, and cohort study). We successfully generated the 3 HAVCR1 coding variants and translated each into a mature KIM-1 protein, which migrated similarly to referent KIM-1 and was expressed at the cell surface (Figure 1a–e). Phagocytic uptake of apoptotic and necrotic cells by HEK293 cells expressing these variants was significantly impaired (Figure 1f and g). As rs6149307 produced the most dysfunctional KIM-1 protein (<10% phagocytic capacity vs. referent), we evaluated the clinical significance of having 2 copies of this variant. We identified 808 deceased donor adult kidney transplants performed from 2008 to 2018. We excluded 163 donors whose stored DNA sample was unusable and 18 recipients who received a simultaneous liver transplant, leaving 627 for genotyping and analysis (Supplementary Figure S1). The observed allele frequency for rs6149307 was 0.61, similar to prior studies,8Nakajima T. Wooding S. Satta Y. et al.Evidence for natural selection in the HAVCR1 gene: high degree of amino-acid variability in the mucin domain of human HAVCR1 protein.Genes Immun. 2005; 6: 398-406Crossref PubMed Scopus (31) Google Scholar,S13,S14 with 242 of 627 donors homozygous for this variant, consistent with Hardy-Weinberg equilibrium (Supplementary Table S1). We compared recipients of these kidneys with controls who received donor kidneys heterozygous or homozygous for referent KIM-1 at this locus. Baseline characteristics were similar between groups (Table 1). A total of 165 of 627 recipients developed DGF (26.3%), but surprisingly, the risk of DGF was not increased in subjects compared with controls (unadjusted relative risk, 1.01; 95% confidence interval, 0.77–1.32). When we adjusted for other clinical factors that were significantly associated with DGF in this cohort, the relative risk was similar (0.97; 95% confidence interval, 0.71–1.28; Table 2). There was also no significant difference in mean 7-day postoperative creatinine between groups.Table 1Baseline characteristics of study populationVariablesSubjectshomozygous for rs6149307Met158_Pro162 del(N = 242)Controlsheterozygous or homozygous for referent(N = 385)aControls were defined as heterozygous or homozygous for referent kidney injury molecular-1 at the considered locus.P valueControlsfor sensitivity analysis (N = 203)bFor the sensitivity analysis, controls were defined as having at least 1 copy of referent kidney injury molecule-1 (H0, N = 203, rows 1–5 in Supplementary Table S3).Recipient variablesAge, yr54.0 (13.7)53.3 (13.7)0.55354.7 (13.1)Female sex, n (%)84 (35)147 (38)0.35480 (40)BMI, kg/m228.0 (5.0)28.0 (6.0)0.28627.5 (5.7)Diabetes, n (%)96 (39)151 (39)0.91182 (40)Cumulative combined PRA, %23.4 (34)22.3 (33)0.68821.8 (34)Preop creat, μmol/L671 (265)696 (271)0.269684 (275)Time on dialysis, median (IQR), d724 (730)804 (765)0.574829 (687)Donor variablesAge, yr43.5 (16.7)43.8 (16.6)0.81745.1 (15.6)Female sex, n (%)82 (33.9)142 (36.9)0.44666 (32.5)BMI, kg/m226.8 (6.7)26.3 (5.5)0.38526.3 (5.0)Hypertension, n (%)67 (29)84 (23)0.11247 (24.0)Diabetes, n (%)18 (7.9)26 (7.1)0.72715 (7.6)Terminal creat, μmol/L76 (50)79 (563)0.50577 (45)ECD, n (%)cECD is defined as donor age ≥60 years and 2 of the following: donor hypertension, terminal creatinine >133 mmol/L, or death from cerebrovascular accident.58 (24)88 (23)0.74950 (25)DCD, n (%)85 (35)129 (34)0.67868 (34)Transplant variablesCold ischemia time, min735 (349)710 (338)0.365697 (321)BMI, body mass index; creat, creatinine; DCD, donor after cardiac death; ECD, extended criteria donor; IQR, interquartile range; PRA, panel-reactive antibody; Preop, preoperative.Data are given as mean (SD), unless otherwise indicated.a Controls were defined as heterozygous or homozygous for referent kidney injury molecular-1 at the considered locus.b For the sensitivity analysis, controls were defined as having at least 1 copy of referent kidney injury molecule-1 (H0, N = 203, rows 1–5 in Supplementary Table S3).c ECD is defined as donor age ≥60 years and 2 of the following: donor hypertension, terminal creatinine >133 mmol/L, or death from cerebrovascular accident. Open table in a new tab Table 2Predictors of delayed graft function in 627 kidney transplant recipientsVariableUnadjusted relative riskaControl group for all analyses is the recipients of donor kidneys with 1 or no copies of the HAVCR1 variant of consideration (N = 385), except as indicated by footnote b.95% CIAdjusted relative riskcAll variables with P < 0.10 in the univariate model were tested in the multivariable model using backward elimination and were retained if P < 0.10, except for HAVCR1 polymorphism, which was retained irrespective of P value. Recipient body mass index, donor sex, and duration of dialysis were removed from the multivariable model as P ≥ 0.10. Bold data refer to those variables included in both adjusted and unadjusted analysis. Dashes indicate that these variables were removed from the multivariable adjusted model at P < 0.10.95% CIHAVCR1 polymorphisms in donor kidneyHomozygous for rs6149307 (N = 242) (main analysis)1.01aControl group for all analyses is the recipients of donor kidneys with 1 or no copies of the HAVCR1 variant of consideration (N = 385), except as indicated by footnote b.0.77–1.320.970.71–1.28Homozygous for rs6149307 (N = 242) (sensitivity analysis)1.03bControl group is the recipients of donor kidneys with at least 1 referent HAVCR1 haplotype (H0, N = 203, rows 1–5 in Supplementary Table S3).0.85–1.25——Other donor factorsDonor age, yr1.000.99–1.01Donor male sex1.131.0001–1.28——Donor body mass index, kg/m21.020.99–1.04Donor hypertension1.250.93–1.63Donor diabetes0.970.57–1.64Donor terminal creatinine, μmol/L1.000.997–1.002Extended criteria donor1.250.92–1.69Donor after cardiac death2.411.97–2.951.991.78–2.15Cold ischemia time, min1.000.999–1.001Recipient factorsRecipient age1.010.997–1.015Recipient male sex1.211.07–1.361.421.15–1.68Recipient body mass index, kg/m21.031.01–1.06——Recipient diabetes1.040.84–1.29Cumulative combined PRA, %1.000.99–1.01Preoperative creatinine, μmol/L1.001.000–1.001Duration of dialysis, mo1.0041.002–1.006——CI, confidence interval; PRA, panel-reactive antibody.a Control group for all analyses is the recipients of donor kidneys with 1 or no copies of the HAVCR1 variant of consideration (N = 385), except as indicated by footnote b.b Control group is the recipients of donor kidneys with at least 1 referent HAVCR1 haplotype (H0, N = 203, rows 1–5 in Supplementary Table S3).c All variables with P < 0.10 in the univariate model were tested in the multivariable model using backward elimination and were retained if P < 0.10, except for HAVCR1 polymorphism, which was retained irrespective of P value. Recipient body mass index, donor sex, and duration of dialysis were removed from the multivariable model as P ≥ 0.10.Bold data refer to those variables included in both adjusted and unadjusted analysis. Dashes indicate that these variables were removed from the multivariable adjusted model at P < 0.10. Open table in a new tab BMI, body mass index; creat, creatinine; DCD, donor after cardiac death; ECD, extended criteria donor; IQR, interquartile range; PRA, panel-reactive antibody; Preop, preoperative. Data are given as mean (SD), unless otherwise indicated. CI, confidence interval; PRA, panel-reactive antibody. The other 2 variants (rs12522248 and rs45439103) that produced modest impairments in phagocytosis in vitro could theoretically have altered kidney susceptibility to IRI. Therefore, we examined the combination of these variants (haplotypes) in our control group (Supplementary Tables S2 and S3). Almost 50% (182 of 385) of controls received donor kidneys with 2 variant KIM-1 haplotypes: 5.5% (35 of 627) were homozygous for rs12522248 or rs45439103, and 23% (147 of 627) were heterozygous for 2 variants on different chromosomes (Supplementary Table S3). More important, when we excluded these individuals and limited our control group to the 203 recipients of donor kidneys having at least 1 referent KIM-1 haplotype, our results did not change (unadjusted relative risk, 1.03; 95% confidence interval, 0.85–1.25; Table 2). When we excluded recipients of kidneys donated after cardiac death (a strong predictor of DGF), we also obtained similar relative risk estimates as in the primary analysis. Herein, we identified 3 nonsynonymous HAVCR1 variants encoding for KIM-1 proteins that impaired phagocytosis of apoptotic and necrotic cells, a process thought to be critically important to mitigate tissue damage and kidney dysfunction after kidney IRI in animal models.2Arai S. Kitada K. Yamazaki T. et al.Apoptosis inhibitor of macrophage protein enhances intraluminal debris clearance and ameliorates acute kidney injury in mice.Nat Med. 2016; 22: 183-193Crossref PubMed Scopus (127) Google Scholar,3Lee J.Y. Ismail O.Z. Zhang X. et al.Donor kidney injury molecule-1 promotes graft recovery by regulating systemic necroinflammation.Am J Transplant. 2018; 18: 2021-2028Crossref PubMed Scopus (11) Google Scholar,9Yang L. Brooks C.R. Xiao S. et al.KIM-1-mediated phagocytosis reduces acute injury to the kidney.J Clin Invest. 2015; 125: 1620-1636Crossref PubMed Scopus (196) Google Scholar We found the most severe variant, rs6149307, which encodes for a 5–amino acid deletion within the mucin domain, nearly abolished phagocytosis. Mice carrying deletion of KIM-1 mucin domain are highly vulnerable to kidney IRI.9Yang L. Brooks C.R. Xiao S. et al.KIM-1-mediated phagocytosis reduces acute injury to the kidney.J Clin Invest. 2015; 125: 1620-1636Crossref PubMed Scopus (196) Google Scholar Yet surprisingly, we observed no increased risk of DGF or differences in 7-day creatinine in recipients who received donor kidneys homozygous for rs6149307, compared with controls. These findings suggest that an aberrant KIM-1 protein, which confers epithelial cells with almost no ability to phagocytose dying cells, is not deleterious in the setting of kidney IRI in humans. Our findings are in direct contrast to those observed in mouse models,2Arai S. Kitada K. Yamazaki T. et al.Apoptosis inhibitor of macrophage protein enhances intraluminal debris clearance and ameliorates acute kidney injury in mice.Nat Med. 2016; 22: 183-193Crossref PubMed Scopus (127) Google Scholar,9Yang L. Brooks C.R. Xiao S. et al.KIM-1-mediated phagocytosis reduces acute injury to the kidney.J Clin Invest. 2015; 125: 1620-1636Crossref PubMed Scopus (196) Google Scholar and have implications for our understanding of kidney IRI, DGF, and its treatment. Warm and cold IRI are inevitable during kidney transplantation. Although most recipients experience no clinically apparent consequences of transplant IRI, 25% to 30% will develop DGF,S15 as we observed. DGF results in increased morbidity and costs,S11 and is associated with short-term rejection and long-term graft loss.S16,S17 Our findings suggest that emerging therapies for DGF and acute kidney injury aimed at enhancing KIM-1 phagocytic function may not be effective in humans. More attention should perhaps be focused on other mechanisms, including those mediated by KIM-1 that do not involve phagocytosis. Others have shown that graft or urinary KIM-1 from human donors does not associate with DGF,5Schroppel B. Kruger B. Walsh L. et al.Tubular expression of KIM-1 does not predict delayed function after transplantation.J Am Soc Nephrol. 2010; 21: 536-542Crossref PubMed Scopus (55) Google Scholar,S18 whereas ours is the first study testing the importance of KIM-1 function and kidney IRI in humans. Differences between human disease and animal models have been observed in countless other settings,S19 and it is possible that murine models are not indicative of human kidney IRI. A more likely explanation is that KIM-1 is protective after kidney IRI via a nonphagocytic mechanism, such as through its inhibition of the heterotrimeric protein, Gα12.7Ismail O.Z. Zhang X. Wei J. et al.Kidney injury molecule-1 protects against Galpha12 activation and tissue damage in renal ischemia-reperfusion injury.Am J Pathol. 2015; 185: 1207-1215Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar We previously showed harmful effects of Gα12 are inhibited by constitutive binding of KIM-1 to Gα12 via its cytosolic domain.7Ismail O.Z. Zhang X. Wei J. et al.Kidney injury molecule-1 protects against Galpha12 activation and tissue damage in renal ischemia-reperfusion injury.Am J Pathol. 2015; 185: 1207-1215Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar,S20 To date, no polymorphisms have been identified within the cytosolic domain that binds KIM-1 to Gα12. A strength of our study is that we empirically measured aberrant function of a human protein in vitro and then correlated this with clinically relevant outcomes. By including consecutive kidney donors at a single center, we were able to employ a cohort design, which is less prone to bias than case-control studies.S21 Our outcome was objective, clinically relevant, and easily measurable, and we were able to capture baseline characteristics with minimal missing data. We are confident in our negative findings as we had adequate statistical power to detect clinically relevant associations between homozygosity for the rs6149307 variant, if such an association exists. Finally, we are reassured that our findings are consistent across sensitivity analyses. We recognize our study's limitations. Because of its retrospective design, we cannot rule out residual confounding from unmeasured factors, including alloimmune responses. We did not have histology to assess tissue injury or inflammation. We also had limited statistical power to analyze the less severe rs12522248 and rs45438103 variants. In summary, to our knowledge, this is the first study to identify functional polymorphisms in a phagocytic receptor in humans and test its relevance to human disease. The role of KIM-1 has been well studied in animal models, but human studies have largely focused on the utility of KIM-1 as a biomarker for tubular epithelial injury rather than on its function. Our results suggest that, contrary to mouse models, severe defects in KIM-1–mediated phagocytosis do not predispose to acute kidney dysfunction after IRI in humans. This work sheds new light on the biology of KIM-1 in humans and may have implications for current therapies aimed at improving DGF and acute kidney injury by targeting KIM-1 function. All the authors declared no competing interests. We thank Samantha Parsons and Jessica House for help with ethics protocol submission and data collection. This work was funded by an Innovation grant from the Academic Medical Organization of Southwestern Ontario and a Canadian National Transplant Research Program Astellas Innovation grant. JYL is the recipient of an Ontario Graduate Scholarship. RSS was supported by a clinician-researcher award from the Fonds de Recherche du Québec–Santé. Download .pdf (.76 MB) Help with pdf files Supplementary File (PDF)